Formulations and methods for treating ulcerative colitis

ABSTRACT

This invention provides composition and methods to prevent, treat or alleviate a symptom of ulcerative colitis comprising administering to a patient dolcanatide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/US2017/013017, filedJan. 11, 2017, which claims the benefit of U.S. Provisional ApplicationNo. 62/277,329 filed Jan. 11, 2016, the contents of each of which areherein incorporated by reference in their entireties for all purposes

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing named“SYPA-015_N01US_SeqList_ST25.txt”. The text file is about 105 kb, wascreated on Jul. 11, 2018, and is being submitted electronically viaEFS-Web.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named “SYPA_015_001WO_SeqList_ST25.txt”,which was recorded on Jan. 11, 2017 and is 105 KB in size, are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the therapeutic use of guanylatecyclase C (GC-C) agonists the treatment of inflammatory bowel disease,such as, ulcerative colitis. The agonists may be used either alone oreither concurrently or sequentially with additional active agents toprevent, treat or alleviate one or more symptoms of ulcerative colitis.

BACKGROUND OF THE INVENTION

Ulcerative colitis (UC) is an inflammatory bowel disease that primarilyaffects the colonic mucosa and sub-mucosa. The most common symptoms ofulcerative colitis are ulcers and inflammation of the inner lining ofthe colon that lead to symptoms of bloody diarrhea, passage of pus,mucus, and abdominal cramping during bowel movements. Currently, thereis no effective therapy to cure the disease. As such, treatment mainlydepends on reduction of the symptoms. Most people with mild or moderateulcerative colitis are treated with corticosteroids (dexamethasone,prednisone, methylprednisone, and hydrocortisone) to reduce inflammationand relieve symptoms. Other drugs typically used to treat symptomsinclude immunomodulators (azathioprine and 6-mercapto-purine) andaminosalicylates. Many of these drugs are associated with numerous sideeffects. Dexamethasone, for example, has many side effects on liver andkidney functions.

A need exists for compositions and methods to treat, prevent andalleviate one or more symptoms of ulcerative colitis without sideeffects

SUMMARY OF THE INVENTION

The invention provides methods for preventing, treating, or alleviatinga symptom of ulcerative colitis by rectally administering to a subjectin need thereof a therapeutically effective amount of a compositioncomprising dolcanatide (i.e., SP-333 or SEQ ID NO: 9). The effectiveamount is between about 1 mg to 10 mg per dose. Preferably, the dose is6 mg. The composition is administered once daily. Preferably, thecomposition is administered once daily for 28 days. The composition isadministered prior to bedtime. The composition is administered as anenema or a suppository. Optionally, the release of the composition istime dependent. Also provided by the invention are formulations forrectal administration comprising the peptide of SEQ ID NO: 9, where saidpeptide is a (4,12:7,15) bicycle. The formulation is a liquid such assaline. Alternatively, the formulation is an enema or a suppository.

In another aspect the invention provides a rectal formulation comprising6 mg of dolcanatide (SEQ ID NO: 9). The formulation is aqueous.

DETAILED DESCRIPTION

It should be understood that singular forms such as “a,” “an,” and “the”are used throughout this application for convenience, however, exceptwhere context or an explicit statement indicates otherwise, the singularforms are intended to include the plural. Further, it should beunderstood that every journal article, patent, patent application,publication, and the like that is mentioned herein is herebyincorporated by reference in its entirety and for all purposes. Allnumerical ranges should be understood to include each and everynumerical point within the numerical range, and should be interpreted asreciting each and every numerical point individually. The endpoints ofall ranges directed to the same component or property are inclusive, andintended to be independently combinable.

“About” includes all values having substantially the same effect, orproviding substantially the same result, as the reference value. Thus,the range encompassed by the term “about” will vary depending on contextin which the term is used, for instance the parameter that the referencevalue is associated with. Thus, depending on context, “about” can mean,for example, ±15%, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±less than 1%.Importantly, all recitations of a reference value preceded by the term“about” are intended to also be a recitation of the reference valuealone.

The present invention is based upon the development of agonists ofguanylate cyclase-C (GC-C). The agonists are analogs of uroguanylin,guanylin, lymphoguanylin and E. coli ST peptide.

In particular the invention is based upon the discovery that rectaldelivery of Dolcanatide (also referred to herein as SP-333 or SEQ IDNO:9) or Plecanatide (also referred to herein as SP-304 or SEQ ID NO:1)to ameliorate the symptoms of ulcerative colitis. Dolcanatide is ananalog of uroguanylin with enhanced resistance to standard digestivebreakdown by proteases in the intestine. It has been previouslydemonstrated in a number of preclinical models the potentialanti-inflammatory role of uroguanylin and uroguanylin analogs incolitis. In these earlier animal studies, oral treatment withdolcanatide was shown to ameliorate DSS- and TNBS-induced acute colitisin murine models. Uroguanylin analog treatment was also shown toameliorate spontaneous colitis in T-cell receptor alpha knockout mice,

A double-blind, placebo-controlled, four-week study evaluating 28patients with mild-to-moderate ulcerative colitis was conducted.Patients received 6 mg Dolcanatide dissolved in saline administeredintra-rectally each evening for 28 days. Analysis of the data indicatesclear signals of improvement in dolcanatide-treated patients comparedwith placebo-treated patients. In particular, dolcanatide-treatedpatients after 28 days of treatment were in remission from theirulcerative colitis symptoms. Dolcanatide was also safe and welltolerated.

Accordingly, the invention provides a method for preventing, treating oralleviating a symptom of ulcerative colitis by rectally administering toa subject in need of a therapeutically effective amount Dolcanatide orPlecanatide. In some embodiments, the subject has mild to moderateulcerative colitis. Symptoms of ulcerative colitis include for example,diarrhea mixed with blood and mucus, and those symptoms recited inTable 1. In some embodiments, ulcerative colitis is scored according toTable 1.

TABLE 1 Ulcerative Colitis Symptom Severity Very Severe Mild ModerateSevere (Fulminant) Up to 4 loose 4-6 loose More than More than 10 loosestools per day stools per day 6 bloody stools per day Stools may Stoolsmay loose stools Constant blood in be bloody be bloody per day stoolsMild Moderate Fever, anemia, Abdominal abdominal pain abdominal pain andrapid tenderness/distention Anemia heart rate Blood transfusion may be arequirement Potentially fatal complications

Also included in the invention are rectal formulations of any of theGC-C agonists represented by Formulas I-XX as well as those amino acidsequences summarized below in Tables 2-8. The GC-C agonists according tothe invention are collectively referred to herein as “GCRA peptides”. Insome embodiments, the GC-C agonist has the sequence of SEQ ID NO: 1(SP-304), SP-333 (SEQ ID NO: 9); SP373 (SEQ ID NO.:104), SP364 (SEQ IDNO.:100), SP366 (SEQ ID NO.:102) or SED ID NO: 250.

In some embodiments, provided GC-C agonist formulations are useful foradministration to a subject undergoing acute opioid use. In someembodiments, provided GC-C agonist formulations are useful foradministration to patients suffering from post-operativegastrointestinal dysfunction, bowel dysfunction, or respiratorydepression.

GCRA Peptides

The GCRA peptides of the present invention are analogues of uroguanylin,guanylin, lymphoguanylin and ST peptides. No particular length isimplied by the term “peptide”. In some embodiments, the GCRA peptide isless than 25 amino acids in length, e.g., less than or equal to 20, 15,14, 13, 12, 11, 10, or 5 amino acids in length.

A preferred GCRA peptide according to the invention is SP-333 (SEQ IDNO:9). In some embodiments, the GCRA peptide is SP-333 (SEQ ID NO: 9),wherein the peptide is a (4,12; 7, 15) bicycle. In some embodiments, theGCRA peptide is SP-304 (SEQ ID NO: 1). In some embodiments, the GCRApeptide is SP-304 (SEQ ID NO: 1), wherein the peptide is a (4,12; 7, 15)bicycle.

The GCRA peptides can be polymers of L-amino acids, D-amino acids, or acombination of both. For example, in various embodiments, the peptidesare D retro-inverso peptides. The term “retro-inverso isomer” refers toan isomer of a linear peptide in which the direction of the sequence isreversed and the chirality of each amino acid residue is inverted. See,e.g., Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature,368, 692-693 (1994). The net result of combining D-enantiomers andreverse synthesis is that the positions of carbonyl and amino groups ineach amide bond are exchanged, while the position of the side-chaingroups at each alpha carbon is preserved. Unless specifically statedotherwise, it is presumed that any given L-amino acid sequence of theinvention may be made into a D retro-inverso peptide by synthesizing areverse of the sequence for the corresponding native L-amino acidsequence. For example a GCRA peptide includes the sequence defined byFormulas I-XX and those listed on Tables 2-8.

By inducing cGMP production is meant that the GCRA peptide induces theproduction of intracellular cGMP. Intracellular cGMP is measured bymethods known in the art. For example, the GCRA peptides of theinvention stimulate 5%, 10%, 20%, 30%, 40%, 50%, 75%, 90% or moreintracellular cGMP compared to naturally occurring GC-C agonists. Infurther embodiments, the GCRA peptides stimulate apoptosis, e.g.,programmed cell death, or activate the cystic fibrosis transmembraneconductance regulator (CFTR).

As used herein PEG3, 3 PEG, is meant to denote polyethylene glycol suchas include aminoethyloxy-ethyloxy-acetic acid (AeeA).

As used herein, the term “AMIDE” is meant to denote that the terminalcarboxylic acid is replaced with an amide group, i.e., the terminal COOHis replaced with CONH₂.

As used herein, the term “pyGlu” refers to pyroglutamic acid.

As used herein, (e.g., in Formulas I-XX) X_(aa) is any natural,unnatural amino acid or amino acid analogue; M_(aa) is a Cysteine (Cys),Penicillamine (Pen) homocysteine, or 3-mercaptoproline. Xaa_(n1) ismeant to denote an amino acid sequence of any natural, unnatural aminoacid or amino acid analogue that is one, two or three residues inlength; Xaa_(n2) is meant to denote an amino acid sequence of anynatural, unnatural amino acid or amino acid analogue that is zero or oneresidue in length; and Xaa_(n3) is meant to denote an amino acidsequence of any natural, unnatural amino acid or amino acid analoguethat is zero, one, two, three, four, five or six residues in length.Additionally, any amino acid represented by Xaa, may be an L-amino acid,a D-amino acid, a methylated amino acid, a fluorinated amino acid or anycombination of thereof. Preferably the amino acids at the N-terminus,C-terminus or both are D-amino acids. Optionally, any GCRA peptiderepresented by Formulas I-XX may contain on or more polyethylene glycolresidues at the N-terminus, C-terminus or both. An exemplarypolyethylene glycol includes aminoethyloxy-ethyloxy-acetic acid andpolymers thereof.

Specific examples of GCC agonist peptides that can be used in themethods and formulations of the invention include a peptide selectedfrom Tables 2-8.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula I, wherein at least one amino acid ofFormula I is a D-amino acid or a methylated amino acid and/or the aminoacid at position 16 is a serine. Preferably, the amino acid at position16 of Formula I is a D-amino acid or a methylated amino acid. Forexample, the amino acid at position 16 of Formula I is a d-leucine or ad-serine. Optionally, one or more of the amino acids at positions 1-3 ofFormula I are D-amino acids or methylated amino acids or a combinationof D-amino acids or methylated amino acids. For example, Asn¹, Asp² orGlu³ (or a combination thereof) of Formula I is a D-amino acid or amethylated amino acid. Preferably, the amino acid at position Xaa⁶ ofFormula I is a leucine, serine or tyrosine.

In alternative embodiments, GCC agonist peptides include peptides havingthe amino acid sequence of Formula II, wherein at least one amino acidof Formula II is a D-amino acid or a methylated amino acid. Preferably,the amino acid denoted by Xaa_(n2) of Formula II is a D-amino acid or amethylated amino acid. In some embodiments, the amino acid denoted byXaa_(n2) of Formula II is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more amino acids denoted by Xaa_(n1) ofFormula II are D-amino acids or methylated amino acids. Preferably, theamino acid at position Xaa⁶ of Formula II is a leucine, a serine, or atyrosine.

In some embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula III, wherein at least one amino acid ofFormula III is a D-amino acid or a methylated amino acid and/or Maa isnot a cysteine. Preferably, the amino acid denoted by Xaa_(n2) ofFormula III is a D-amino acid or a methylated amino acid. In someembodiments the amino acid denoted by Xaa_(n2) of Formula III is aleucine, a d-leucine, a serine, or a d-serine. Preferably, the one ormore amino acids denoted by Xaa_(n1) of Formula III are D-amino acids ormethylated amino acids. Preferably, the amino acid at position Xaa⁶ ofFormula III is a leucine, a serine, or a tyrosine.

In other embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula IV, wherein at least one amino acid ofFormula IV is a D-amino acid or a methylated amino acid, and/or Maa isnot a cysteine. Preferably, the Xaa_(n2) of Formula IV is a D-amino acidor a methylated amino acid. In some embodiments, the amino acid denotedby Xaa_(n2) of Formula IV is a leucine, a d-leucine, a serine, or ad-serine. Preferably, the one or more of the amino acids denoted byXaa_(n1) of Formula IV are D-amino acids or methylated amino acids.Preferably, the amino acid denoted Xaa⁶ of Formula IV is a leucine, aserine, or a tyrosine.

In further embodiments, GCC agonist peptides include peptides having theamino acid sequence of Formula V, wherein at least one amino acid ofFormula V is a D-amino acid or a methylated amino acid. Preferably, theamino acid at position 16 of Formula V is a D-amino acid or a methylatedamino acid. For example, the amino acid at position 16 (i.e., Xaa¹⁶) ofFormula V is a d-leucine or a d-serine. Optionally, one or more of theamino acids at position 1-3 of Formula V are D-amino acids or methylatedamino acids or a combination of D-amino acids or methylated amino acids.For example, Asn¹, Asp² or Glu³ (or a combination thereof) of Formula Vis a D-amino acids or a methylated amino acid. Preferably, the aminoacid denoted at Xaa⁶ of Formula V is a leucine, a serine, or a tyrosine.

In additional embodiments, GCRA peptides include peptides having theamino acid sequence of Formula VI, VII-a, VII-b, VIII, or IX.Preferably, the amino acid at position 6 of Formula VI, VII-a, VII-b,VIII, or IX is a leucine, a serine or a tyrosine. In some aspects theamino acid at position 16 of Formula VI, VII-a, VII-b, VIII or IX is aleucine or a serine. Preferably, the amino acid at position 16 ofFormula VI, VII-a, VII-b, VIII or IX is a D-amino acid or a methylatedamino acid.

In additional embodiments, GCRA peptides include peptides having theamino acid sequence of Formula X, XI, XII, XIII, XIV, XV, XVI or XVII.Optionally, one or more amino acids of Formulas X, XI, XII, XIII, XIV,XV, XVI or XVII are D-amino acids or methylated amino acids. Preferably,the amino acid at the carboxyl terminus of the peptides according toFormulas X, XI, XII, XIII, XIV, XV, XVI or XVII is a D-amino acid or amethylated amino acid. For example the amino acid at the carboxylterminus of the peptides according to Formulas X, XI, XII, XIII, XIV,XV, XVI or XVII is a D-tyrosine.

Preferably, the amino acid denoted by Xaa⁶ of Formula XIV is a tyrosine,phenylalanine or a serine. Most preferably the amino acid denoted byXaa⁶ of Formula XIV is a phenylalanine or a serine. Preferably, theamino acid denoted by Xaa⁴ of Formula XV, XVI or XVII is a tyrosine, aphenylalanine, or a serine. Most preferably, the amino acid positionXaa⁴ of Formula XV, XVI or XVII is a phenylalanine or a serine.

In some embodiments, GCRA peptides include peptides containing the aminoacid sequence of Formula XVIII. Preferably, the amino acid at position 1of Formula XVIII is a glutamic acid, aspartic acid, glutamine or lysine.Preferably, the amino acid at position 2 and 3 of Formula XVIII is aglutamic acid, or an aspartic acid. Preferably, the amino acid atposition 5 is a glutamic acid. Preferably, the amino acid at position 6of Formula XVIII is an isoleucine, valine, serine, threonine ortyrosine. Preferably, the amino acid at position 8 of Formula XVIII is avaline or isoleucine. Preferably, the amino acid at position 9 ofFormula XVIII is an asparagine. Preferably, the amino acid at position10 of Formula XVIII is a valine or a methionine. Preferably, the aminoacid at position 11 of Formula XVIII is an alanine. Preferably, theamino acid at position 13 of Formula XVIII is a threonine. Preferably,the amino acid at position 14 of Formula XVIII is a glycine. Preferably,the amino acid at position 16 of Formula XVIII is a leucine, serine orthreonine

In alternative embodiments, GCRA peptides include peptides containingthe amino acid sequence of Formula XIX. Preferably, the amino acid atposition 1 of Formula XIX is a serine or asparagine. Preferably, theamino acid at position 2 of Formula XIX is a histidine or an asparticacid. Preferably, the amino acid at position 3 of Formula XIX is athreonine or a glutamic acid. Preferably, the amino acid at position 5of Formula XIX is a glutamic acid. Preferably, the amino acid atposition 6 of Formula XIX is an isoleucine, leucine, valine or tyrosine.Preferably, the amino acid at position 8, 10, 11, or 13 of Formula XIXis an alanine. Preferably, the amino acid at position 9 of Formula XIXis an asparagine or a phenylalanine. Preferably, the amino acid atposition 14 of Formula XIX is a glycine.

In further embodiments, GCRA peptides include peptides containing theamino acid sequence of Formula XX. Preferably, the amino acid atposition 1 of Formula XX is a glutamine. Preferably, the amino acid atposition 2 or 3 of Formula XX is a glutamic acid or an aspartic acid.Preferably, the amino acid at position 5 of Formula XX is a glutamicacid. Preferably, the amino acid at position 6 of Formula XX isthreonine, glutamine, tyrosine, isoleucine, or leucine. Preferably, theamino acid at position 8 of Formula XX is isoleucine or valine.Preferably, the amino acid at position 9 of Formula XX is asparagine.Preferably, the amino acid at position 10 of Formula XX is methionine orvaline. Preferably, the amino acid at position 11 of Formula XX isalanine. Preferably, the amino acid at position 13 of Formula XX is athreonine. Preferably, the amino acid at position 1 of Formula XX is aglycine. Preferably, the amino acid at position 15 of Formula XX is atyrosine. Optionally, the amino acid at position 15 of Formula XX istwo-amino acid in length and is Cysteine (Cys), Penicillamine (Pen)homocysteine, or 3-mercaptoproline and serine, leucine or threonine.

In certain embodiments, one or more amino acids of the GCRA peptides canbe replaced by a non-naturally occurring amino acid or a naturally ornon-naturally occurring amino acid analog. There are many amino acidsbeyond the standard 20 (Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val). Some arenaturally-occurring others are not. (See, for example, Hunt, TheNon-Protein Amino Acids: In Chemistry and Biochemistry of the AminoAcids, Barrett, Chapman and Hall, 1985). For example, an aromatic aminoacid can be replaced by 3,4-dihydroxy-L-phenylalanine,3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) ornor-tyrosine (norTyr). Phg and norTyr and other amino acids includingPhe and Tyr can be substituted by, e.g., a halogen, —CH3, —OH, —CH2NH3,—C(O)H, —CH2CH3, —CN, —CH2CH2CH3, —SH, or another group. Any amino acidcan be substituted by the D-form of the amino acid.

With regard to non-naturally occurring amino acids or naturally andnon-naturally occurring amino acid analogs, a number of substitutions inthe polypeptide and agonists described herein are possible alone or incombination.

For example, glutamine residues can be substituted withgamma-Hydroxy-Glu or gamma-Carboxy-Glu. Tyrosine residues can besubstituted with an alpha substituted amino acid such asL-alpha-methylphenylalanine or by analogues such as: 3-Amino-Tyr;Tyr(CH3); Tyr(PO3(CH3)2); Tyr(SO3H); beta-Cyclohexyl-Ala;beta-(1-Cyclopentenyl)-Ala; beta-Cyclopentyl-Ala; beta-Cyclopropyl-Ala;beta-Quinolyl-Ala; beta-(2-Thiazolyl)-Ala; beta-(Triazole-1-yl)-Ala;beta-(2-Pyridyl)-Ala; beta-(3-Pyridyl)-Ala; Amino-Phe; Fluoro-Phe;Cyclohexyl-Gly; tBu-Gly; beta-(3-benzothienyl)-Ala;beta-(2-thienyl)-Ala; 5-Methyl-Trp; and A-Methyl-Trp. Proline residuescan be substituted with homopro (L-pipecolic acid); hydroxy-Pro;3,4-Dehydro-Pro; 4-fluoro-Pro; or alpha-methyl-Pro or anN(alpha)-C(alpha) cyclized amino acid analogues with the structure: n=0,1, 2, 3 Alanine residues can be substituted with alpha-substituted orN-methylated amino acid such as alpha-amino isobutyric acid (aib),L/D-alpha-ethylalanine (L/D-isovaline), L/D-methylvaline, orL/D-alpha-methylleucine or a non-natural amino acid such asbeta-fluoro-Ala. Alanine can also be substituted with: n=0, 1, 2, 3Glycine residues can be substituted with alpha-amino isobutyric acid(aib) or L/D-alpha-ethylalanine (L/D-isovaline).

Further examples of unnatural amino acids include: an unnatural analogof alanine (e.g., L-1-Nal or L-2-Nal); an unnatural analog of tyrosine;an unnatural analogue of glutamine; an unnatural analogue ofphenylalanine; an unnatural analogue of serine; an unnatural analogue ofthreonine; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine,hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno,ester, thioacid, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or aminosubstituted amino acid, or any combination thereof; an amino acid with aphotoactivatable cross-linker; a spin-labeled amino acid; a fluorescentamino acid; an amino acid with a novel functional group; an amino acidthat covalently or noncovalently interacts with another molecule; ametal binding amino acid; an amino acid that is amidated at a site thatis not naturally amidated, a metal-containing amino acid; a radioactiveamino acid; a photocaged and/or photoisomerizable amino acid; a biotinor biotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid (e.g., an amino acid containing deuterium, tritium, ¹³C, ¹⁵N,or ¹⁸O); a chemically cleavable or photocleavable amino acid; an aminoacid with an elongated side chain; an amino acid containing a toxicgroup; a sugar substituted amino acid, e.g., a sugar substituted serineor the like; a carbon-linked sugar-containing amino acid; a redox-activeamino acid; an α-hydroxy containing acid; an amino thio acid containingamino acid; an α, α disubstituted amino acid; a β-amino acid; a cyclicamino acid other than proline; an O-methyl-L-tyrosine; anL-3-(2-naphthyl)alanine; a 3-methyl-phenylalanine; aρ-acetyl-L-phenylalanine; an O-4-allyl-L-tyrosine; a4-propyl-L-tyrosine; a tri-O-acetyl-GlcNAcβ-serine; an L-Dopa; afluorinated phenylalanine; an isopropyl-L-phenylalanine; ap-azido-L-phenylalanine; a p-acyl-L-phenylalanine; ap-benzoyl-L-phenylalanine; an L-phosphoserine; a phosphonoserine; aphosphonotyrosine; a p-iodo-phenylalanine; a 4-fluorophenylglycine; ap-bromophenylalanine; a p-amino-L-phenylalanine; anisopropyl-L-phenylalanine; L-3-(2-naphthyl)alanine;D-3-(2-naphthyl)alanine (dNal); an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue; a dopa, 0-methyl-L-tyrosine;a glycosylated amino acid; a p-(propargyloxy)phenylalanine;dimethyl-Lysine; hydroxy-proline; mercaptopropionic acid; methyl-lysine;3-nitro-tyrosine; norleucine; pyro-glutamic acid; Z (Carbobenzoxyl);ε-Acetyl-Lysine; β-alanine; β-aspartic acid; β-cyclohexylalanine;aminobenzoyl derivative; aminobutyric acid (Abu); citrulline;aminohexanoic acid (Ahx); aminoisobutyric acid (AIB); cyclohexylalanine;d-cyclohexylalanine; cyclohexylglycine; hydroxyproline; nitro-arginine;nitro-phenylalanine; nitro-tyrosine; norvaline; octahydroindolecarboxylate; ornithine (Orn); penicillamine (PEN);tetrahydroisoquinoline; diaminobutyric acid; diaminopimelic acid;pyroglutamic acid; homocysteine; homoserine; N-ε-dinitrophenyl-lysine;N-ε-methyl-lysine; N-ε-dimethyl-lysine; N,N,N-ε-trimethyl-lysine;acetamidomethyl protected amino acids and pegylated amino acids. Furtherexamples of unnatural amino acids and amino acid analogs can be found inU.S. 20030108885, U.S. 20030082575, US20060019347 (paragraphs 410-418)and the references cited therein. The polypeptides of the invention caninclude further modifications including those described inUS20060019347, paragraph 589.

“Nal” used herein refers to both L-1-naphthylalanine (L-1-Nal) andL-2-naphthylalanine (L-2-Nal).

In some embodiments, an amino acid can be replaced by anaturally-occurring, non-essential amino acid, e.g., taurine.

Alternatively, the GCRA peptides are cyclic peptides. GCRA cyclicpeptides are prepared by methods known in the art. For example,macrocyclization is often accomplished by forming an amide bond betweenthe peptide N- and C-termini, between a side chain and the N- orC-terminus [e.g., with K₃Fe(CN)6 at pH 8.5] (Samson et al.,Endocrinology, 137: 5182-5185 (1996)), or between two amino acid sidechains, such as cysteine. See, e.g., DeGrado, Adv Protein Chem, 39:51-124 (1988). In some embodiments, the GCRA peptides of the presentinvention are bicyclic peptides. In various aspects the GCRA peptidesare [4,12; 7,15] bicycles.

In some GCRA peptides one or both members of one or both pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, penicillamine, 3-mercaptoproline (Kolodziej et al. 1996Int J Pept Protein Res 48:274); β, β dimethylcysteine (Hunt et al. 1993Int JPept Protein Res 42:249) or diaminopropionic acid (Smith et al.1978 J Med Chem 2 1:117) to form alternative internal cross-links at thepositions of the normal disulfide bonds.

In addition, one or more disulfide bonds can be replaced by alternativecovalent cross-links, e.g., an amide linkage (—CH2CH(O)NHCH2- or—CH2NHCH(O)CH2-), an ester linkage, a thioester linkage, a lactambridge, a carbamoyl linkage, a urea linkage, a thiourea linkage, aphosphonate ester linkage, an alkyl linkage (—CH2CH2CH2CH2-), an alkenyllinkage(—CH 2CH═CHCH 2-), an ether linkage (—CH2CH2OCH2- or—CH2OCH2CH2-), a thioether linkage (—CH2CH2SCH2- or —CH2SCH2CH2-), anamine linkage (—CH2CH2NHCH2- or —CH2NHCH 2CH2-) or a thioamide linkage(—CH2CH(S)HNHCH 2- or —CH2NHCH(S)CH2-). For example, Ledu et al. (ProcNat'l Acad. Sci. 100:11263-78, 2003) describe methods for preparinglactam and amide cross-links. Exemplary GCRA peptides which include alactam bridge include for example SP-370.

The GCRA peptides can have one or more conventional polypeptide bondsreplaced by an alternative bond. Such replacements can increase thestability of the polypeptide. For example, replacement of thepolypeptide bond between a residue amino terminal to an aromatic residue(e.g. Tyr, Phe, Trp) with an alternative bond can reduce cleavage bycarboxy peptidases and may increase half-life in the digestive tract.Bonds that can replace polypeptide bonds include: a retro-inverso bond(C(O)—NH instead of NH—C(O); a reduced amide bond (NH—CH2); athiomethylene bond (S—CH2 or CH2-S); an oxomethylene bond (O—CH2 orCH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)—NH); atrans-olefine bond (CH═CH); a fluoro substituted trans-olefme bond(CF═CH); a ketomethylene bond (C(O)—CHR or CHR—C(O) wherein R is H orCH3; and a fluoro-ketomethylene bond (C(O)—CFR or CFR—C(O) wherein R isH or F or CH3.

The GCRA peptides can be modified using standard modifications.Modifications may occur at the amino (N—), carboxy (C—) terminus,internally or a combination of any of the preceding. In one aspectdescribed herein, there may be more than one type of modification on thepolypeptide. Modifications include but are not limited to: acetylation,amidation, biotinylation, cinnamoylation, farnesylation, formylation,myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr),stearoylation, succinylation, sulfurylation and cyclisation (viadisulfide bridges or amide cyclisation), and modification by Cys3 orCys5. The GCRA peptides described herein may also be modified by 2,4-dinitrophenyl (DNP), DNP-lysine, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine). The GCRApeptides described herein may also be conjugated to, for example,polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight orbranched alkyl groups); fatty acid radicals; combinations of PEG, alkylgroups and fatty acid radicals (See, U.S. Pat. No. 6,309,633; Soltero etal., 2001 Innovations in Pharmaceutical Technology 106-110); BSA and KLH(Keyhole Limpet Hemocyanin). The addition of PEG and other polymerswhich can be used to modify polypeptides of the invention is describedin US2006019347 section IX.

In one aspect, the invention provides an Aad-GCRA peptide as taught in,for example, WO20140151206 which is hereby incorporated by reference inits entirety for all purposes. Table 1 of WO20140151206 disclosesexamples of various alpha-aminoadipic acid derivatives of GCRA Peptides.The Aad-GCRA peptides are analogues uroguanylin, guanylin,lymphoguanylin and ST peptides. Particularly, these analogs contain anα-aminoadipic acid (Ad), preferably at the 3rd position from theN-terminus of each peptide or at the position to the N-terminal sidenext to the first cysteine (“Cys) residue. For example an Aad-GCRApeptide includes the sequences defined by Formulae I, II, III, IV, V,VI, VII, VII, VIII, IX, XVIII or XXI which can comprise an α-aminoadipicacid.

In some embodiments, the Aad-GCRA peptide isAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶(SEQ ID NO: 258; SP-304-Aad),dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶(SEQ ID NO: 253; SP-333-Aad);Pyglu¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶(SEQ ID NO: 254; SP-373-Aad),dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶(SEQ ID NO: 255; SP-364-Aad);dAsn¹-Asp²-Aad³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dTyr¹⁶(SEQ ID NO: 256; SP-366-Aad); orXaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Xaa_(n2)¹⁶ (SEQ ID NO: 257).

Also included in the invention are peptides that biologically orfunctional equivalent to the peptides described herein. The term“biologically equivalent” or functional equivalent” is intended to meanthat the compositions of the present invention are capable ofdemonstrating some or all of the cGMP production modulatory effects.

GCRA peptides can also include derivatives of GCRA peptides which areintended to include hybrid and modified forms of GCRA peptides in whichcertain amino acids have been deleted or replaced and modifications suchas where one or more amino acids have been changed to a modified aminoacid or unusual amino acid and modifications such as glycosylation solong the modified form retains the biological activity of GCRA peptides.By retaining the biological activity, it is meant that cGMP and orapoptosis is induced by the GCRA peptide, although not necessarily atthe same level of potency as that of a naturally-occurring GCRA peptideidentified.

Preferred variants are those that have conservative amino acidsubstitutions made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in a GCRApolypeptide is replaced with another amino acid residue from the sameside chain family. Alternatively, in another embodiment, mutations canbe introduced randomly along all or part of a GCRA coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedto identify mutants that retain activity.

TABLE 2 GCRA Peptides (SP-304 and Derivatives) SEQ Position of ID NameDisulfide bonds Structure NO SP-304 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶  1 SP-326 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴-Leu¹⁵  2 SP-327 C3:C11, C6:C14Asp¹-Glu²-Cys³-Glu⁴-Leu⁵-Cys⁶-Val⁷-Asn⁸-Val⁹-Ala¹⁰-Cys¹¹-Thr¹²-Gly¹³-Cys¹⁴  3 SP-328 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Leu¹⁴  4 SP-329 C2:C10, C5:C13Glu¹-Cys²-Glu³-Leu⁴-Cys⁵-Val⁶-Asn⁷-Val⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³   5SP-330 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²-Leu¹³   6SP-331 C1:C9, C4:C12Cys¹-Glu²-Leu³-Cys⁴-Val⁵-Asn⁶-Val⁷-Ala⁸-Cys⁹-Thr¹⁰-Gly¹¹-Cys¹²   7 SP332C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  8 SP-333 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶  9 SP-334 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 10 SP-335 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 11 SP-336 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Leu¹⁶ 12 SP-337 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-dLeu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 13 SP-338 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵ 14 SP-342 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 15 SP-343 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 16 SP-344 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 17 SP-347 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 18 SP-348 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 19 SP-350 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 20 SP-352 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 21 SP-358 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶- 22 PEG3 SP-359 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 23 SP-360 C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 24 SP-361 C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 25 SP-362 C4:C12, C7:C15PEG3-dAsn¹-dAsp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 26 SP-368 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dNal¹⁶ 27 SP-369 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-AIB⁸-Asn⁹-AIB¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 28 SP-370 C4:C12, 7:15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Asp[Lactam]⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Orn¹⁵-dLeu¹ 29 SP-371 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 30 SP-372 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 31 N1 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 32 N2 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 33 N3 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 34 N4 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 35 N5 C4:C12, C7:C15PEG3-dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶ 36 N6 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dLeu¹⁶-PEG3 37 N7 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 38 N8 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 39 N9 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶ 40 N10 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-Ser¹⁶-PEG3 41 N11 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 42 N12 C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶ 43 N13 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-dSer¹⁶-PEG3 44 Formula I C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Aaa¹¹-Cys¹²-Aaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 45 Formula II C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa_(n2) 46 Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Glu⁵-Xaa⁶-Maa⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Maa¹²-Thr¹³-Gly¹⁴-Maa¹⁵-Xaa_(n2) 47 III Formula 4:12, 7:15Xaa_(n1)-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Maa¹⁵-Xaa_(n2) 48 IV Formula V C4:C12, C7:C15Asn¹-Asp²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-Xaa¹⁶ 49 Formula C4:C12, C7:C15dAsn¹-Glu²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 50 VI Formula C4:C12, C7:C15dAsn¹-dGlu²-Asp³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 51 VII-a Formula C4:C12, C7:C15dAsn¹-dAsp²-Glu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Asn⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 52 VII-b Formula C4:C12, C7:C15dAsn¹-dAsp²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 53 VIII Formula C4:C12, C7:C15dAsn¹-dGlu²-dGlu³-Cys⁴-Xaa⁵-Xaa⁶-Cys⁷-Xaa⁸-Tyr⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-d-Xaa¹⁶ 54 IX Formula C4:C12, C7:C15Xaa_(n1)-Cys⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Xaa¹⁵-Xaa_(n2)¹⁶ 250 XXI

TABLE 3 Linaclotide and Derivatives Position of SEQ ID NameDisulfide Bonds Structure NO: SP-339 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴55 (linaclotide) SP-340 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 56SP-349 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-57 PEG3 SP-353 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-58 Tyr¹⁶ SP-354 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-59 Tyr¹⁶ SP-355 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-dTyr¹⁴60 SP-357 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴61 SP-374 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-62 Tyr¹⁶ SP-375 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-63 dTyr¹⁶ SP-376 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-64 Tyr¹⁶ SP-377 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-65 dTyr¹⁶ SP-378 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-66 dTyr¹⁶ SP-379 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-67 Tyr¹⁶ SP-380 C3:C8, C4:C12, C7:C15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-68 dTyr¹⁶ SP-381 C3:C8, C4:C12, C7:15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-69 dTyr¹⁶ SP-382 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-70 Tyr¹⁶ SP-383 C3:C8, C4:C12, C7:15dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-71 dTyr¹⁶ SP384 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴-PEG372 N14 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG373 N15 C1:C6, C2:C10, C5:C13PEG3-Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³74 N16 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Tyr⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-PEG375 N17 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-76 Cys¹⁵-Tyr¹⁶-PEG3 N18 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-77 Cys¹⁵-Tyr¹⁶ N19 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Ser⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-78 Tyr¹⁶-PEG3 N20 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-79 Cys¹⁵-Tyr¹⁶-PEG3 N21 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-80 Cys¹⁵-Tyr¹⁶ N22 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Phe⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-81 Tyr¹⁶-PEG3 N23 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-82 Cys¹⁵-Tyr¹⁶-PEG3 N24 C3:C8, C4:C12, C7:C15PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-83 Cys¹⁵-Tyr¹⁶ N25 C3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-84 Tyr¹⁶-PEG3 N26 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴85 N27 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³-Tyr¹⁴86 N28 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Ser⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³- 87N29 C1:C6, C2:C10, C5:C13Cys¹-Cys²-Glu³-Phe⁴-Cys⁵-Cys⁶-Asn⁷-Pro⁸-Ala⁹-Cys¹⁰-Thr¹¹-Gly¹²-Cys¹³ 88N30 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³-Tyr¹⁴89 N31 1:6, 2:10, 5:13Pen¹-Pen²-Glu³-Tyr⁴-Pen⁵-Pen⁶-Asn⁷-Pro⁸-Ala⁹-Pen¹⁰-Thr¹¹-Gly¹²-Pen¹³ 90Formula X C9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Tyr¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-91 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIC9:C14, C10:C18,Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Asn⁷-Phe⁸-Cys⁹-Cys¹⁰-Xaa¹¹-Phe¹²-Cys¹³-Cys¹⁴-Xaa¹⁵-92 C13:C21 Xaa¹⁶-Xaa¹⁷-Cys¹⁸-Xaa¹⁹-Xaa²⁰-Cys²¹-Xaa²² Formula XIIC3:C8, C4:C12, C7:C15Asn¹-Phe²-Cys³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Cys⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-93 Xaa¹⁶ Formula XIII 3:8, 4:12, 7:15Asn¹-Phe²-Pen³-Cys⁴-Xaa⁵-Phe⁶-Cys⁷-Pen⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Cys¹²-Xaa¹³-Xaa¹⁴-Cys¹⁵-94 Xaa¹⁶ Formula XIV 3:8, 4:12, 7:15Asn¹-Phe²-Maa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Maa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-95 Maa¹⁵-Xaa¹⁶ Formula XV 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³-Tyr¹⁴96 Formula XVI 1:6, 2:10, 5:13Maa¹-Maa²-Glu³-Xaa⁴-Maa⁵-Maa⁶-Asn⁷-Pro⁸-Ala⁹-Maa¹⁰-Thr¹¹-Gly¹²-Maa¹³ 97Formula XVII 1:6, 2:10, 5:13Xaa_(n3)-Maa¹-Maa²-Xaa³-Xaa⁴-Maa⁵-Maa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Maa¹⁰-Xaa¹¹-Xaa¹²-Maa¹³-98 Xaa_(n2)

TABLE 4 GCRA Peptides Position of SEQ ID  Name Disulfide bonds StructureNO: SP-363 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³- 99 Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ SP-364 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-100 Gly¹⁴-Cys¹⁵-dSer¹⁶ SP-365 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-101 Gly¹⁴-Cys¹⁵-dSer-AMIDE¹⁶ SP-366 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-102 Gly¹⁴-Cys¹⁵-dTyr¹⁶ SP-367 C4:C12, C7:C15dAsn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-103 Gly¹⁴-Cys¹⁵-dTyr-AMIDE¹⁶ SP-373 C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-104 Gly¹⁴-Cys¹⁵-dLeu-AMIDE¹⁶ / C4:C12, C7:C15Pyglu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-251 Gly¹⁴-Cys¹⁵-Leu¹⁶ SP- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-105 304diPEG Gly¹⁴-Cys¹⁵-Leu¹⁶-PEG3 SP-304N- C4:C12, C7:C15PEG3-Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-106 PEG Gly¹⁴-Cys¹⁵-Leu¹⁶ SP-304C- C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-107 PEG Cys¹⁵-Leu¹⁶-PEG3

TABLE 5 SP-304 Analogs, Uroguanylin, and Uroguanylin Analogs Position ofSEQ ID Name Disulfide bonds Structure NO Formula C4:C12,Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-108 XVIII C7:C15 Xaa¹⁴-Maa¹⁵-Xaa¹⁶ Uro- C4:C12,Asn¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-109 guanylin C7:C15 Gly¹⁴-Cys¹⁵-Leu¹⁶ N32 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-110 Gly¹⁴-Cys¹⁵-Leu¹⁶ N33 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-111 Gly¹⁴-Cys¹⁵-Leu¹⁶ N34 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-112 Gly¹⁴-Cys¹⁵-Leu¹⁶ N35 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-113 Gly¹⁴-Cys¹⁵-Leu¹⁶ N36 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-114 Gly¹⁴-Cys¹⁵-Leu¹⁶ N37 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-115 Gly¹⁴-Cys¹⁵-Leu¹⁶ N38 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-116 Gly¹⁴-Cys¹⁵-Leu¹⁶ N39 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-117 Gly¹⁴-Cys¹⁵-Leu¹⁶ N40 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-118 Gly¹⁴-Cys¹⁵-Leu¹⁶ N41 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-119 Gly¹⁴-Cys¹⁵-Leu¹⁶ N42 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-120 Gly¹⁴-Cys¹⁵-Leu¹⁶ N43 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-121 Gly¹⁴-Cys¹⁵-Leu¹⁶ N44 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-122 Gly¹⁴-Cys¹⁵-Leu¹⁶ N45 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-123 Gly¹⁴-Cys¹⁵-Leu¹⁶ N46 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-124 Gly¹⁴-Cys¹⁵-Leu¹⁶ N47 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-125 Gly¹⁴-Cys¹⁵-Leu¹⁶ N48 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-126 Gly¹⁴-Cys¹⁵-Leu¹⁶ N49 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-127 Gly¹⁴-Cys¹⁵-Leu¹⁶ N50 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-128 Gly¹⁴-Cys¹⁵-Leu¹⁶ N51 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-129 Gly¹⁴-Cys¹⁵-Leu¹⁶ N52 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-⁶130 Gly¹⁴-Cys¹⁵-Leu¹ N53 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-131 Gly¹⁴-Cys¹⁵-Leu¹⁶ N54 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-132 Gly¹⁴-Cys¹⁵-Leu¹⁶ N55 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-133 Gly¹⁴-Cys¹⁵-Leu¹⁶ N56 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-134 Gly¹⁴-Cys¹⁵-Leu¹⁶ N57 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-135 Gly¹⁴-Cys¹⁵-Leu¹⁶ N58 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-136 Gly¹⁴-Cys¹⁵-Leu¹⁶ N59 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-137 Gly¹⁴-Cys¹⁵-Leu¹⁶ N60 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-138 Gly¹⁴-Cys¹⁵-Leu¹⁶ N61 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-139 Gly¹⁴-Cys¹⁵-Leu¹⁶ N62 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-140 Gly¹⁴-Cys¹⁵-Leu¹⁶ N63 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Val⁸-Asn⁹-Val¹⁰-Ala¹¹-Cys¹²-Thr¹³-141 Gly¹⁴-Cys¹⁵-Leu¹⁶ N65 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-142 Gly¹⁴-Cys¹⁵-Leu¹⁶ N66 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-143 Gly¹⁴-Cys¹⁵-Leu¹⁶ N67 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-144 Gly¹⁴-Cys¹⁵-Leu¹⁶ N68 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-145 Gly¹⁴-Cys¹⁵-Leu¹⁶ N69 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-146 Gly¹⁴-Cys¹⁵-Leu¹⁶ N70 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-147 Gly¹⁴-Cys¹⁵-Leu¹⁶ N71 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-148 Gly¹⁴-Cys¹⁵-Leu¹⁶ N72 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-149 Gly¹⁴-Cys¹⁵-Leu¹⁶ N73 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-150 Gly¹⁴-Cys¹⁵-Leu¹⁶ N74 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-151 Gly¹⁴-Cys¹⁵-Leu¹⁶ N75 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-152 Gly¹⁴-Cys¹⁵-Leu¹⁶ N76 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-153 Gly¹⁴-Cys¹⁵-Leu¹⁶ N77 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-154 Gly¹⁴-Cys¹⁵-Leu¹⁶ N78 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-155 Gly¹⁴-Cys¹⁵-Leu¹⁶ N79 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-156 Gly¹⁴-Cys¹⁵-Leu¹⁶ N80 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-157 Gly¹⁴-Cys¹⁵-Leu¹⁶ N81 C4:C12, C7:C15Glu¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-158 Gly¹⁴-Cys¹⁵-Leu¹⁶ N82 C4:C12, C7:C15Glu¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-159 Gly¹⁴-Cys¹⁵-Leu¹⁶ N83 C4:C12, C7:C15Glu¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-160 Gly¹⁴-Cys¹⁵-Leu¹⁶ N84 C4:C12, C7:C15Glu¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-161 Gly¹⁴-Cys¹⁵-Leu¹⁶ N85 C4:C12, C7:C15Asp¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-162 Gly¹⁴-Cys¹⁵-Leu¹⁶ N86 C4:C12, C7:C15Asp¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-163 Gly¹⁴-Cys¹⁵-Leu¹⁶ N87 C4:C12, C7:C15Asp¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-164 Gly¹⁴-Cys¹⁵-Leu¹⁶ N88 C4:C12, C7:C15Asp¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-165 Gly¹⁴-Cys¹⁵-Leu¹⁶ N89 C4:C12, C7:C15Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-166 Gly¹⁴-Cys¹⁵-Leu¹⁶ N90 C4:C12, C7:C15Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-167 Gly¹⁴-Cys¹⁵-Leu¹⁶ N91 C4:C12, C7:C15Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-168 Gly¹⁴-Cys¹⁵-Leu¹⁶ N92 C4:C12, C7:C15Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-169 Gly¹⁴-Cys¹⁵-Leu¹⁶ N93 C4:C12, C7:C15Lys¹-Asp²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-170 Gly¹⁴-Cys¹⁵-Leu¹⁶ N94 C4:C12, C7:C15Lys¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-171 Gly¹⁴-Cys¹⁵-Leu¹⁶ N95 C4:C12, C7:C15Lys¹-Glu²-Asp³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-172 Gly¹⁴-Cys¹⁵-Leu¹⁶ N96 C4:C12, C7:C15Lys¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-173 Gly¹⁴-Cys¹⁵-Leu¹⁶

TABLE 6 Guanylin and Analogs Position of SEQ Disulfide ID Name bondsStructure NO Formula 4:12, 7:15Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaaa⁶-Maa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-174 XIX Maa¹⁵ Guanylin C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Phe⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-175 Cys¹⁵ Human C4:C12, C7:C15Pro¹-Gly²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Tyr⁹-Ala¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-252 Guanylin Cys¹⁵ N97 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵176 N98 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵177 N99 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵178 N100 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵179 N101 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵180 N102 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵181 N103 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵182 N104 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵183 N105 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵184 N106 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵185 N107 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵186 N108 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵187 N109 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵188 N110 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵189 N111 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵190 N112 C4:C12, C7:C15Ser¹-His²-Thr³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵191 N113 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵192 N114 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵193 N115 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵194 N116 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵195 N117 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵196 N118 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵197 N119 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵198 N120 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵199 N121 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵200 N122 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵201 N123 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵202 N124 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵203 N125 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵204 N126 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵205 N127 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Val⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵206 N128 C4:C12, C7:C15Asn¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ala⁸-Asn⁹-Ala¹⁰-Ala¹¹-Cys¹²-Ala¹³-Gly¹⁴-Cys¹⁵207

TABLE 7 Lymphoguanylin and Analogs Position of SEQ Disulfide ID Namebonds Structure NO FormulaXX 4:12Xaa¹-Xaa²-Xaa³-Maa⁴-Xaa⁵-Xaa⁶-Maa⁷-Xaa⁸-Xaa⁹-xaa¹⁰-Xaa¹¹-Maa¹²-Xaa¹³-Xaa¹⁴-Xaa_(n1)¹⁵ 208 Lympho- C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Leu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵209 guanylin N129 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵210 N130 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵211 N131 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵212 N132 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵213 N133 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵214 N134 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵215 N135 C4:C12Glni-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵216 N136 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵217 N137 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵218 N138 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵219 N139 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵220 N140 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵221 N141 C4:C12Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵222 N142 C4:C12Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵223 N143 C4:C12Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵224 N144 C4:C12Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Tyr¹⁵225 N145 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-226 C7:C15 Ser¹⁶ N146 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-227 C7:C15 Ser¹⁶ N147 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-228 C7:C15 Ser¹⁶ N148 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-229 C7:C15 Ser¹⁶ N149 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-230 C7:C15 Ser¹⁶ N150 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-231 C7:C15 Ser¹⁶ N151 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-232 C7:C15 Ser¹⁶ N152 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Glu⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-233 C7:C15 Ser¹⁶ N153 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-234 C7:C15 Ser¹⁶ N154 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-235 C7:C15 Ser¹⁶ N155 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-236 C7:C15 Ser¹⁶ N156 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-237 C7:C15 Ser¹⁶ N157 C4:C12,Gln¹-Glu²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-238 C7:C15 Ser¹⁶ N158 C4:C12,Gln¹-Asp²-Glu³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-239 C7:C15 Ser¹⁶ N159 C4:C12,Gln¹-Asp²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-240 C7:C15 Ser¹⁶ N160 C4:C12,Gln¹-Glu²-Asp³-Cys⁴-Glu⁵-Ile⁶-Cys⁷-Ile⁸-Asn⁹-Met¹⁰-Ala¹¹-Cys¹²-Thr¹³-Gly¹⁴-Cys¹⁵-241 C7:C15 Ser¹⁶

TABLE 8 ST Peptide and Analogues Position of SEQ ID Name Disulfide bondsStructure NO STPeptide C9:C14, C10:C18,Asn¹-Ser²-Ser³-Asn⁴-Ser⁵-Ser⁶-Asn⁷-Tyr⁸-Cys⁹-Cys¹⁰-Glu¹¹-Lys¹²-Cys¹³-242 C13:C21 Cys¹⁴-Asn¹⁵-Pro¹⁶-Ala¹⁷-Cys¹⁸-Thr¹⁹-Gly²⁰-Cys²¹-Tyr²² N161C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 243C7:C15 Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 N162 C3:C8, C4:C12,PEG3-Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²- 244C7:C15 Thr¹³-Gly¹⁴-Cys¹⁵-Tyr¹⁶ N163 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Thr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-245 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶-PEG3 N164 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-246 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶ N165 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-247 C7:C15 Gly¹⁴-Cys¹⁵-dTyr¹⁶ N166 C3:C8, C4:C12,Asn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-248 C7:C15 Gly¹⁴-Cys¹⁵-dTyr¹⁶ N167 C3:C8, C4:C12,dAsn¹-Phe²-Cys³-Cys⁴-Glu⁵-Tyr⁶-Cys⁷-Cys⁸-Asn⁹-Pro¹⁰-Ala¹¹-Cys¹²-Thr¹³-249 C7:C15 Gly¹⁴-Cys¹⁵-Tyr¹⁶

The term “consisting essentially of” includes peptides that areidentical to a recited sequence (any one from Tables 2-8) and othersequences that do not differ substantially in terms of either structureor function. For the purpose of the present application, a peptidediffers substantially if its structure varies by more than three aminoacids from a peptide of any one from Tables 2-8 or if its activation ofcellular cGMP production is reduced or enhanced by more than 50%.Preferably, substantially similar peptides should differ by no more thantwo amino acids and not differ by more than about 25% with respect toactivating cGMP production.

Also included within the meaning of substantially homologous is any GCRApeptide which may be isolated by virtue of cross-reactivity withantibodies to the GCRA peptide.

Preparation of GCRA Peptides

GCRA peptides are easily prepared using modern cloning techniques, ormay be synthesized by solid state methods or by site-directedmutagenesis. A GCRA peptide may include dominant negative forms of apolypeptide. Exemplary methods for preparing the GCRA peptide includedthe methods disclosed in WO/2012/118972 and WO/2013/138352, the contentsof which are hereby incorporate by prefer in their entireties.

Chemical synthesis may generally be performed using standard solutionphase or solid phase peptide synthesis techniques, in which a peptidelinkage occurs through the direct condensation of the amino group of oneamino acid with the carboxy group of the other amino acid with theelimination of a water molecule. Peptide bond synthesis by directcondensation, as formulated above, requires suppression of the reactivecharacter of the amino group of the first and of the carboxyl group ofthe second amino acid. The masking substituents must permit their readyremoval, without inducing breakdown of the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (See, Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1-4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor. Solidphase peptide synthesis uses an insoluble polymer for support duringorganic synthesis. The polymer-supported peptide chain permits the useof simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc., 1963, 85:2149, which involves assembling a linearpeptide chain on a resin support using protected amino acids. Solidphase peptide synthesis typically utilizes either the Boc or Fmocstrategy, which is well known in the art.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the synthesis.In addition, solid phase synthesis is generally most suitable whenpeptides are to be made on a small scale.

Acetylation of the N-terminal can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation is accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Alternatively the GCRA peptides are produced by modern cloningtechniques. For example, the GCRA peptides are produced either inbacteria including, without limitation, E. coli, or in other existingsystems for polypeptide or protein production (e.g., Bacillus subtilis,baculovirus expression systems using Drosophila Sf9 cells, yeast orfilamentous fungal expression systems, mammalian cell expressionsystems), or they can be chemically synthesized. If the GCRA peptide orvariant peptide is to be produced in bacteria, e.g., E. coli, thenucleic acid molecule encoding the polypeptide may also encode a leadersequence that permits the secretion of the mature polypeptide from thecell. Thus, the sequence encoding the polypeptide can include the presequence and the pro sequence of, for example, a naturally-occurringbacterial ST polypeptide. The secreted, mature polypeptide can bepurified from the culture medium.

The sequence encoding a GCRA peptide described herein can be insertedinto a vector capable of delivering and maintaining the nucleic acidmolecule in a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof.

Construction of a vector containing a nucleic acid described herein canbe followed by transformation of a host cell such as a bacterium.Suitable bacterial hosts include but are not limited to, E. coli, Bsubtilis, Pseudomonas, Salmonella. The genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences.

A variety of transcriptional control sequences are well known to thosein the art. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, or an internal ribosome entry site). The vector can becapable of autonomous replication or it can integrate into host DNA toensure stability during polypeptide production.

The protein coding sequence that includes a GCRA peptide describedherein can also be fused to a nucleic acid encoding a polypeptideaffinity tag, e.g., glutathione S-transferase (GST), maltose E bindingprotein, protein A, FLAG tag, hexa-histidine, myc tag or the influenzaHA tag, in order to facilitate purification. The affinity tag orreporter fusion joins the reading frame of the polypeptide of interestto the reading frame of the gene encoding the affinity tag such that atranslational fusion is generated. Expression of the fusion gene resultsin translation of a single polypeptide that includes both thepolypeptide of interest and the affinity tag. In some instances whereaffinity tags are utilized, DNA sequence encoding a protease recognitionsite will be fused between the reading frames for the affinity tag andthe polypeptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the GCRA peptides and variants described herein inprotein expression systems other than bacteria, and well known to thoseskilled in the art, can also be used to produce polypeptides in abiological system.

The peptides disclosed herein may be modified by attachment of a secondmolecule that confers a desired property upon the peptide, such asincreased half-life in the body, for example, pegylation. Suchmodifications also fall within the scope of the term “variant” as usedherein.

By “inhibiting” or “inhibition” or “reduce”, it means the GCRA peptidedecreases the activity and/or production of a protein by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 100%, or morerelative to the activity and/or production of the protein without theGCRA peptide.

By “induce”, it means the GCRA peptide increases the activity and/orproduction of a protein by at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100%, or more relative to the activity and/orproduction of the protein without the GCRA peptide.

The term “treatment” or “treating” refers to reducing or alleviatingsymptoms in a subject, preventing symptoms from worsening orprogressing, and/or preventing disease in a subject who is freetherefrom. For a given subject, improvement, worsening, regression, orprogression of a symptom may be determined by any objective orsubjective measure. Efficacy of the treatment may be measured as animprovement in morbidity or mortality (e.g., lengthening of survivalcurve for a selected population). Thus, effective treatment wouldinclude therapy of existing disease, control of disease by slowing orstopping its progression, prevention of disease occurrence, reduction inthe number or severity of symptoms, or a combination thereof. The effectmay be shown in a controlled study using one or more statisticallysignificant criteria.

The term “prevention” in relation to a given disease or disorder means:preventing the onset of disease development if none had occurred,preventing the disease or disorder from occurring in a subject that maybe predisposed to the disorder or disease but has not yet been diagnosedas having the disorder or disease, and/or preventing furtherdisease/disorder development if already present.

Intracellular cGMP produced by exposing, e.g., contacting a tissue(e.g., gastrointestinal tissue; lung tissue, esophageal tissue) or cellwith GCRA agonists. By inducing is meant an increase in cGMP productioncompared to a tissue or cell that has not been in contact with GCRApeptide or variant. Tissues or cells are directly contacted with a GCRApeptide or variant. Alternatively, the GCRA peptide or variant isadministered systemically. GCRA peptide or variant are administered inan amount sufficient to increase intracellular cGMP concentration. cGMPproduction is measured by a cell-based assay known in the ar.

Disorders are treated, prevented or alleviated by administering to asubject, e.g., a mammal such as a human in need thereof, atherapeutically effective dose of a GCRA peptide. The GCRA peptides maybe in a pharmaceutical composition in unit dose form, together with oneor more pharmaceutically acceptable excipients. The term “unit doseform” refers to a single drug delivery entity, e.g., a tablet, capsule,solution or inhalation formulation. The amount of peptide present shouldbe sufficient to have a positive therapeutic effect when administered toa patient. Typically a therapeutically effective amount is a unit doseform between 0.1 mg to 10 mg. Preferably the unit dose form is 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 mg. More preferably the unit dose is 3 mg or 6mg. Most preferably the unit dose for is 6 mg. What constitutes a“positive therapeutic effect” will depend upon the particular conditionbeing treated and will include any significant improvement in acondition readily recognized by one of skill in the art.

The GCRA peptides can be administered alone or in combination withanother therapeutic agent, for example, those to treat, prevent, orameliorate symptoms of inflammatory bowel disease, e.g., ulcerativecolitis. Exemplary therapeutic agents, include but are not limitedbudesonide, iclosporin, tacrolimu, fexofenadine 5-ASA drugs such assulfasalazine and mesalazine, corticosteroids such as prednisone,immunosuppressive medications such as azathioprine and biological agentssuch as infliximab and adalimumab.

Alternatively, two or more different GCRA peptides are administered. Forexample, SP-333 is administered with SEQ ID NO: 1 (SP-304), SP373 (SEQID NO.:104), SP364 (SEQ ID NO.:100), SP366 (SEQ ID NO.:102) or SED IDNO: 250. Preferably, SP-333 is administered rectally. Also for example,SP-304 is administered with SEQ ID NO: 9 (SP-333), SP373 (SEQ IDNO.:104), SP364 (SEQ ID NO.:100), SP366 (SEQ ID NO.:102) or SED ID NO:250. Preferably, SP-304 is administered rectally.

The term “combination therapy” means administering two or more activeagents concurrently or sequentially. Concurrent administration may beachieved with a formulation in which two or more active agents aremixed, or with simultaneous administration of two or more active agentsformulated independently. Sequential administration of two or moreactive agents may be achieved with two or more active agents, formulatedindependently, administered in sequence with one agent administeredfirst followed by the second agent administered seconds, minutes, hours,or days after the first agent.

Combination therapy can be achieved by administering two or more agents,e.g., a GCRA peptide described herein or a composition described hereinand another compound, each of which is formulated and administeredseparately, or by administering two or more agents in a singleformulation. Other combinations are also encompassed by combinationtherapy. For example, two agents can be formulated together andadministered in conjunction with a separate formulation containing athird agent. While the two or more agents in the combination therapy canbe administered simultaneously, they need not be. For example,administration of a first agent (or combination of agents) can precedeadministration of a second agent (or combination of agents) by minutes,hours, days, or weeks. Thus, the two or more agents can be administeredwithin minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 daysof each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of eachother. In some cases even longer intervals are possible. While in manycases it is desirable that the two or more agents used in a combinationtherapy be present in within the patient's body at the same time, thisneed not be so.

The GCRA peptides described herein may be combined withphosphodiesterase inhibitors, e.g., sulindae sulfone, Zaprinast,sildenafil, vardenafil or tadalafil to further enhance levels of cGMP inthe target tissues or organs.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

Combination therapy can also include the administration of two or moreagents via different routes or locations. For example, the GCRA peptide,preferably SP-333 is administered rectally and the other agent isadministered orally, subcutaneously or intravenously. Alternatively,both the GCRA peptide and the other agent are administered rectally. Ineach case, the agents can either simultaneously or sequentially.Approximated dosages for some of the combination therapy agentsdescribed herein are found in the “BNF Recommended Dose” column oftables on pages 11-17 of WO01/76632 (the data in the tables beingattributed to the March 2000 British National Formulary) and can also befound in other standard formularies and other drug prescribingdirectories. For some drugs, the customary prescribed dose for anindication will vary somewhat from country to country.

The GCRA peptides, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium. Thus, they can becombined with materials that do not produce an adverse, allergic orotherwise unwanted reaction when administered to a patient. The carriersor mediums used can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose (e.g. celphere, Celphere Beads®), diluents,lubricants, binders, disintegrating agents, and the like), etc. Ifdesired, tablet dosages of the disclosed compositions may be coated bystandard aqueous or nonaqueous techniques.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include: sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a GCRA agonist) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, methods ofpreparation are vacuum drying and freeze-drying that yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. Such as mannitol, fructooligosaccharides, polyethylene glycoland other excipients. They can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

In some embodiments, the compounds are administered rectally. Drugsadministered rectally generally have a faster onset, higherbioavailability, shorter peak, and shorter duration than drugsadministered orally. Rectally administered drugs also reach thecirculatory system with less alteration and in greater concentrationsthereby increasing drug efficacy.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery. In someembodiments, the compounds are prepared in an aqueous formulation. Insome embodiments, the compounds are prepared in a liquid formulation. Insome embodiments, the compounds are prepared in saline.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated fully herein by reference.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, glidants,anti-adherents, anti-static agents, surfactants (wetting agents),anti-oxidants, film-coating agents, and the like. Any such optionalingredient must be compatible with the compound described herein toinsure the stability of the formulation.

The composition may contain other additives as needed, including forexample lactose, glucose, fructose, galactose, trehalose, sucrose,maltose, raffnose, maltitol, melezitose, stachyose, lactitol,palatinite, starch, xylitol, mannitol, myoinositol, and the like, andhydrates thereof, and amino acids, for example alanine, glycine andbetaine, and polypeptides and proteins, for example albumen.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents such as: BINDERS: corn starch, potato starch, otherstarches, gelatin, natural and synthetic gums such as acacia, xanthan,sodium alginate, alginic acid, other alginates, powdered tragacanth,guar gum, cellulose and its derivatives (e.g., ethyl cellulose,cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose), polyvinyl pyrrolidone (e.g., povidone, crospovidone,copovidone, etc), methyl cellulose, Methocel, pre-gelatinized starch(e.g., STARCH 1500® and STARCH 1500 LM®, sold by Colorcon, Ltd.),hydroxypropyl methyl cellulose, microcrystalline cellulose (FMCCorporation, Marcus Hook, Pa., USA), or mixtures thereof, FILLERS: talc,calcium carbonate (e.g., granules or powder), dibasic calcium phosphate,tribasic calcium phosphate, calcium sulfate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,dextrose, fructose, honey, lactose anhydrate, lactose monohydrate,lactose and aspartame, lactose and cellulose, lactose andmicrocrystalline cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose &amp; guar gum, molasses, sucrose, ormixtures thereof, DISINTEGRANTS: agar-agar, alginic acid, calciumcarbonate, microcrystalline cellulose, croscarmellose sodium,crospovidone, polacrilin potassium, sodium starch glycolate, potato ortapioca starch, other starches, pre-gelatinized starch, clays, otheralgins, other celluloses, gums (like gellan), low-substitutedhydroxypropyl cellulose, or mixtures thereof, LUBRICANTS: calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, sodium stearyl fumarate, vegetable based fattyacids lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil,cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil andsoybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloidsilica gel (AEROSIL 200, W.R. Grace Co., Baltimore, Md. USA), acoagulated aerosol of synthetic silica (Deaussa Co., Piano, Tex. USA), apyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA), ormixtures thereof, ANTI-CAKING AGENTS: calcium silicate, magnesiumsilicate, silicon dioxide, colloidal silicon dioxide, talc, or mixturesthereof, ANTIMICROBIAL AGENTS: benzalkonium chloride, benzethoniumchloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridiniumchloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben,methylparaben, phenol, phenylethyl alcohol, phenoxyethanol,phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate,propylparaben, sodium benzoate, sodium dehydroacetate, sodiumpropionate, sorbic acid, thimersol, thymo, or mixtures thereof, andCOATING AGENTS: sodium carboxymethyl cellulose, cellulose acetatephthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropylmethyl cellulose phthalate, methylcellulose, polyethylene glycol,polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide,carnauba wax, microcrystalline wax, gellan gum, maltodextrin,methacrylates, microcrystalline cellulose and carrageenan or mixturesthereof.

The formulation can also include other excipients and categories thereofincluding but not limited to L-histidine, Pluronic®, Poloxamers (such asLutrol® and Poloxamer 188), ascorbic acid, glutathione, permeabilityenhancers (e.g. lipids, sodium cholate, acylcarnitine, salicylates,mixed bile salts, fatty acid micelles, chelators, fatty acid,surfactants, medium chain glycerides), protease inhibitors (e.g. soybeantrypsin inhibitor, organic acids), pH lowering agents and absorptionenhancers effective to promote bioavailability (including but notlimited to those described in U.S. Pat. Nos. 6,086,918 and 5,912,014),creams and lotions (like maltodextrin and carrageenans); materials forchewable tablets (like dextrose, fructose, lactose monohydrate, lactoseand aspartame, lactose and cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose and guar gum, sorbitol crystalline);parenterals (like mannitol and povidone); plasticizers (like dibutylsebacate, plasticizers for coatings, polyvinylacetate phthalate); powderlubricants (like glyceryl behenate); soft gelatin capsules (likesorbitol special solution); spheres for coating (like sugar spheres);spheronization agents (like glyceryl behenate and microcrystallinecellulose); suspending/gelling agents (like carrageenan, gellan gum,mannitol, microcrystalline cellulose, povidone, sodium starch glycolate,xanthan gum); sweeteners (like aspartame, aspartame and lactose,dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses,sorbitol crystalline, sorbitol special solution, sucrose); wetgranulation agents (like calcium carbonate, lactose anhydrous, lactosemonohydrate, maltodextrin, mannitol, microcrystalline cellulose,povidone, starch), caramel, carboxymethylcellulose sodium, cherry creamflavor and cherry flavor, citric acid anhydrous, citric acid,confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake,disodium edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake,FD&C Blue #1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminumlake, FD&C Green No. 3, FD&C Red No. 40, FD&C Yellow No. 6 AluminumLake, FD&C Yellow No. 6, FD&C Yellow No. 10, glycerol palmitostearate,glyceryl monostearate, indigo carmine, lecithin, manitol, methyl andpropyl parabens, mono ammonium glycyrrhizinate, natural and artificialorange flavor, pharmaceutical glaze, poloxamer 188, Polydextrose,polysorbate 20, polysorbate 80, polyvidone, pregelatinized corn starch,pregelatinized starch, red iron oxide, saccharin sodium, sodiumcarboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate,strawberry flavor, synthetic black iron oxide, synthetic red iron oxide,titanium dioxide, and white wax.

Solid oral dosage forms may optionally be treated with coating systems(e.g. Opadry® fx film coating system, for example Opadry® blue(OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white (YS-1-7040), andblack ink (S-1-8 106).

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycoloic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. 3,773,919), polylactic acid (U.S. 4,767,628),poly(ε-caprolactone) and poly(alkylene oxide) (U.S. 20030068384) tocreate a sustained release formulation. Such formulations can be used toimplants that release a polypeptide or another agent over a period of afew days, a few weeks or several months depending on the polymer, theparticle size of the polymer, and the size of the implant (See, e.g.,U.S. Pat. No. 6,620,422). Other sustained release formulations andpolymers for use in are described in EP 0 467 389 A2, WO 93/24150, U.S.Pat. No. 5,612,052, WO 97/40085, WO 03/075887, WO 01/01964A2, U.S. Pat.No. 5,922,356, WO 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat. Nos.5,968,895, 6,180,608, U.S. 20030171296, U.S. 20020176841, U.S. Pat. Nos.5,672,659, 5,893,985, 5,134,122, 5,192,741, 5,192,741, 4,668,506,4,713,244, 5,445,832, 4,931,279, 5,980,945, WO 02/058672, WO 97/26015,WO 97/04744, and US20020019446. In such sustained release formulationsmicroparticles (Delie and Blanco-Prieto 2005 Molecule 10:65-80) ofpolypeptide are combined with microparticles of polymer. One or moresustained release implants can be placed in the large intestine, thesmall intestine or both. U.S. Pat. No. 6,011,011 and WO 94/06452describe a sustained release formulation providing either polyethyleneglycols (i.e. PEG 300 and PEG 400) or triacetin. WO 03/053401 describesa formulation which may both enhance bioavailability and providecontrolled releaseof the agent within the GI tract. Additionalcontrolled release formulations are described in WO 02/38129, EP 326151,U.S. Pat. No. 5,236,704, WO 02/30398, WO 98/13029; U.S. 20030064105,U.S. 20030138488A1, U.S. 20030216307A1, U.S. Pat. No. 6,667,060, WO01/49249, WO 01/49311, WO 01/49249, WO 01/49311, and U.S. Pat. No.5,877,224 materials which may include those described in WO04041195(including the seal and enteric coating described therein) andpH-sensitive coatings that achieve delivery in the colon including thosedescribed in U.S. Pat. No. 4,910,021 and W9001329. U.S. Pat. No.4,910,021 describes using a pH-sensitive material to coat a capsule.WO9001329 describes using pH-sensitive coatings on beads containingacid, where the acid in the bead core prolongs dissolution of thepH-sensitive coating. U.S. Pat. No. 5,175,003 discloses a dual mechanismpolymer mixture composed of pH-sensitive enteric materials andfilm-forming plasticizers capable of conferring permeability to theenteric material, for use in drug-delivery systems; a matrix pelletcomposed of a dual mechanism polymer mixture permeated with a drug andsometimes covering a pharmaceutically neutral nucleus; a membrane-coatedpellet comprising a matrix pellet coated with a dual mechanism polymermixture envelope of the same or different composition; and apharmaceutical dosage form containing matrix pellets. The matrix pelletreleases acid-soluble drugs by diffusion in acid pH and bydisintegration at pH levels of nominally about 5.0 or higher.

In some embodiments, the compositions described herein are formulated ina pH dependent release form. Alternatively, such compositions areformulated in a form that releases the peptides at a specific region ofthe gastrointestinal (GI) tract (e.g., duodenum, jejunum, ileum,terminal ileum, or ascending colon). The formulation may contain aninert carrier coated with a composition and an enteric coating whichreleases the peptides at a specific pH (such as pH5 or pH7). PreferredpH for duodenum or jejunum release is pH 4.5-5.5 or pH 5.5-6.5.Preferred pH for ileum, terminal ileum, or ascending colon release is pH5.5-6.5 or pH 6.5-7.5. Preferably, the inert carrier is a selected frommannitol, lactose, a microcrystalline cellulose, or starch.

The GCRA peptides described herein may be formulated in the pH triggeredtargeted control release systems described in WO04052339. The agentsdescribed herein may be formulated according to the methodologydescribed in any of WO03105812 (extruded hyrdratable polymers);WO0243767 (enzyme cleavable membrane translocators); WO03007913 andWO03086297 (mucoadhesive systems); WO02072075 (bilayer laminatedformulation comprising pH lowering agent and absorption enhancer);WO04064769 (amidated polypeptides); WO05063156 (solid lipid suspensionwith pseudotropic and/or thixotropic properties upon melting);WO03035029 and WO03035041 (erodible, gastric retentive dosage forms);US5007790 and US5972389 (sustained release dosage forms); WO041 1271 1(oral extended release compositions); WO05027878, WO02072033, andWO02072034 (delayed release compositions with natural or synthetic gum);WO05030182 (controlled release formulations with an ascending rate ofrelease); WO05048998 (microencapsulation system); U.S. Pat. No.5,952,314 (biopolymer); U.S. Pat. No. 5,108,758 (glassy amylose matrixdelivery); U.S. Pat. No. 5,840,860 (modified starch based delivery).JP10324642 (delivery system comprising chitosan and gastric resistantmaterial such as wheat gliadin or zein); U.S. Pat. Nos. 5,866,619 and6,368,629 (saccharide containing polymer); U.S. Pat. No. 6,531,152(describes a drug delivery system containing a water soluble core (Capectinate or other water-insoluble polymers) and outer coat which bursts(e.g. hydrophobic polymer-Eudragrit)); U.S. Pat. Nos. 6,234,464;6,403,130 (coating with polymer containing casein and high methoxypectin; WO0174 175 (Maillard reaction product); WO05063206 (solubilityincreasing formulation); WO040 19872 (transferring fusion proteins).

Rectal Formulations

In preferred embodiments, the GCC agonists are formulated for rectaldelivery. More preferably SP-333 or SP-304 is formulated for rectaldelivery. For example, the GCC agonists are administered rectally bymeans of an enema, a suppository, rectal catheters or liposomally. Insome embodiments, the release of the composition is time dependent.

In particular, compositions for rectal administration include solutionsand emulsions.

The compositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Preferred aqueoussolutions include saline. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. In some embodiments, theformulation is an aqueous formulation. In some embodiments, theformulation is a liquid formulation. In some embodiments, the liquid issaline.

In some embodiments, the formulations for rectal administration comprisethe peptide of SEQ ID NO: 9, where said peptide is a (4,12:7,15)bicycle. In some embodiments, the formulations for rectal administrationcomprise the peptide of SEQ ID NO: 1, where said peptide is a(4,12:7,15) bicycle. In some embodiments, the formulation is a liquidsuch as saline. In some embodiments, formulation is aqueous. In someembodiments the formulations for rectal administration comprise 6 mg ofdolcanatide (SEQ ID NO: 9).

In some embodiments, the formulation is an enema or a suppository.Formulations for rectal administration may be presented as asuppository, which may be prepared by mixing the GCC agonists,preferably SP-333 with one or more suitable non-irritating carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax, or a salicylate, and which is solid at roomtemperature, but liquid at body temperature and, therefore, will melt inthe appropriate body cavity and release the encapsulated compound(s) andcomposition(s). suppositories, and enemas.

Dosage

Dosage levels of active ingredients in a pharmaceutical composition canalso be varied so as to achieve a transient or sustained concentrationof the compound in a subject, especially in and around the site ofinflammation or disease area, and to result in the desired response. Itis well within the skill of the art to start doses of the compound atlevels lower than required to achieve the desired effect and togradually increase the dosage until the desired effect is achieved. Itwill be understood that the specific dose level for any particularsubject will depend on a variety of factors, including body weight,general health, diet, natural history of disease, route and schedulingof administration, combination with one or more other drugs, andseverity of disease.

An effective dosage of the composition will typically be between 0.1 mgto 10 mg. Adjustments in dosage will be made using methods that areroutine in the art and will be based upon the particular compositionbeing used and clinical considerations.

Dosage levels of the GCR agonist for use in methods of this inventiontypically are from about 0.1 mg to about 10 mg daily. For example, aneffective dosage of the GCRA peptide e.g. SP-333 or SP-304) for use inmethods of this invention is about 0.1 to 100 mg per day, or optionallytwice a day. In some embodiments, an effective dosage of the CGRApeptide (e.g. SP-333 or SP-304) for use in methods of this invention isabout 0.1, about 0.2. about 0.3, about 0.4.about 0.5, about 0.6, about0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5,about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0,about 9.5, about 10, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, or about 100 mg per day, or optionallytwice a day. Preferably the GCRA peptide is given after in the evening,such as after dinner or before bedtime. In some embodiments, the GCRApeptide is SP-333 and is administered at a dose of about 2 mg to about 8mg. In some embodiments, the GCRA peptide is SP-333 and is administeredat a dose of about 2.7 mg, about 3 mg, about 5.7 mg, or about 6 mg. Insome embodiments, the GCRA peptide is SP-304 and is administered at adose of about 2 mg to about 10 mg. In some embodiments, the GCRA peptideis SP-304 and is administered at a dose of about 3 mg, about 6 mg, orabout 9 mg.

In some embodiments, the GCR agonist is administered weekly, daily, ormulti-daily. In preferred embodiments, the GCR agonist is administereddaily.

The GCRA peptide can be administered until ulcerative colitis symptomsimprove. In some embodiments, the GCRA peptide is administered for about1 day to about 10 years. In some embodiments, the CGRA peptide isadministered for about 1 day, about 2 days, about 3 days, about 4 days,about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks,about 4 weeks, about 1 month, about 2 months, about 3 months, about 4months, about 5 months, about 6 months, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months, about1 year, about 2 years, about 5 years or longer. In some embodiments, theGCRA peptide is administered for 28 days.

The GCRA peptide can be administered once daily until ulcerative colitissymptoms improve. In some embodiments, the GCRA peptide is administeredonce daily for about 1 day to about 10 years. In some embodiments, theCGRA peptide is administered for about 1 day, about 2 days, about 3days, about 4 days, about 5 days, about 6 days, about 7 days, about 2weeks, about 3 weeks, about 4 weeks, about 1 month, about 2 months,about 3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, about 12 months, about 1 year, about 2 years, about 5 years orlonger. In some embodiments, the GCRA peptide is administered once dailyfor 28 days. In some embodiments SP-333 is administered once daily for28 days.

The total daily dose of each inhibitor can be administered to thepatient in a single dose, or in multiple subdoses. Preferably the totaldaily dose is administered as a single dose. Typically, subdoses can beadministered two to six times per day, preferably two to four times perday, and even more preferably two to three times per day. Doses can bein immediate release form or sustained release form sufficientlyeffective to obtain the desired control over the medical condition.

The dosage regimen to prevent, treat, give relief from, or ameliorate amedical condition or disorder, or to otherwise protect against or treata medical condition with the combinations and compositions of thepresent invention is selected in accordance with a variety of factors.These factors include, but are not limited to, the type, age, weight,sex, diet, and medical condition of the subject, the severity of thedisease, the route of administration, pharmacological considerationssuch as the activity, efficacy, pharmacokinetics and toxicology profilesof the particular inhibitors employed, whether a drug delivery system isutilized, and whether the inhibitors are administered with other activeingredients. Thus, the dosage regimen actually employed may vary widelyand therefore deviate from the preferred dosage regimen set forth above.

EXAMPLES Example 1 Rectal Delivery of Dolcanatide Improved UlcerativeColitis Symptoms

A double-blind, placebo-controlled, four week study was performedevaluating 28 patients with mild to moderate ulcerative colitis.Subjects were treated with either saline (placebo) or Dolcanatide(SP-333; SEQ ID NO: 9). Six mg of Dolcanatide was dissolved in salineand administered intra-rectally every evening for 28 days. At the end oftreatment, dolcanatide-treated patients exhibited clear signals ofimprovement compared with placebo-treated patients. Particularly,dolcanatide-treated patients were in remission from their ulcerativecolitis symptoms after treatment for 28 days.

Example 2 Rectal Delivery of Plecanatide Improved Ulcerative ColitisSymptoms

Subjects with mild-to-moderate ulcerative colitis will receiveintra-rectal administration of either saline or 3 mg, 6 mg, or 9 mgPlecanatide (SP-304; SEQ ID NO: 1) for at least 28 days. At the end oftreatment, subjects will be evaluated for presence and severity ofulcerative colitis symptoms in Plecanatide-treated subjects comparedwith placebo-treated subjects.

We claim:
 1. A method for preventing, treating, or alleviating a symptomof ulcerative colitis, the method comprising rectally administering oncedaily for at least 28 days to a subject in need thereof atherapeutically effective amount of a composition comprising 6 mg ofdolcanatide (SEQ ID NO: 9), and inducing remission of the symptoms ofulcerative colitis in the subject.
 2. The method of claim 1, wherein thecomposition is administered prior to bedtime.
 3. The method of claim 1,wherein the composition is an enema.
 4. The method of claim 1, whereinthe composition is a suppository.
 5. The method of claim 1, wherein therelease of the composition is time dependent.
 6. The method of claim 1,wherein the composition is a liquid.
 7. The method of claim 6, whereinthe liquid is saline.